Fixed mounted sorting cuvette with user replaceable nozzle

ABSTRACT

A flow cell and flow cytometer in which a nozzle at the end of a flow channel is disposed on a removable substrate held at a registered location on a flow cell. Other elements including illumination optics, light collection optics, and the flow cell may then be positioned at fixed locations and would not require subsequent periodic adjustment. The registered location for positioning the nozzle allows removal and replacement of the nozzle key with the nozzle subsequently positioned in the identical location.

TECHNICAL FIELD

[0001] The present invention relates to flow cytometry.

BACKGROUND OF THE INVENTION

[0002] Flow analysis has proven to be an important technology for theanalysis of discrete targets. The applications of this technologyinclude cellular assay to investigate a variety of cellular featuresincluding DNA content, specific nucleic acid sequences, chromaticstructure, RNA content, specific antigens, surface receptors, cellmorphology, DNA degredation and other assay targets. The targets of aflow cytometer may be multicellular organisms (e.g. microfilaria),cellular aggregates, viable cells, dead cells, cell fragments,organelles, large molecules (e.g. DNA), particles such as beads, viralparticles or other discrete targets of this size range. The term“cells”, as used throughout, is used to refer to such discrete targets.This technology has a number of different applications, includingdiagnostic, clinical and research applications.

[0003] Flow cytometry measures targets flowing through an analyticalregion in a flow cell. In the flow cell a core stream is injected intothe center of a sheath flow stream flowing at a constant flow rate. Thecore stream is a liquid sample, which may be injected from a sampletube. Injection generally requires insertion of an aspiration tube intothe sample tube and pressurization of the head above the liquid in thesample tube such that sample liquid is pressure driven from the sampletube into the injection tube.

[0004] The flow stream is directed into a tapered portion of the flowcell body and through an analytical region. In one design, the stream isdirected through a nozzle and analyzed in air. In a second design, thestream is directed through a channel for analysis.

[0005] Analysis takes place by optical interrogation of particles aseach particle passes a detection region. In most systems, one or morelaser beams are directed by steering mirrors and illumination lensesthrough the analytical region. If more than one laser are used, adichroic stack may be used to combine the beams and direct the beamsthrough the stream to be analyzed.

[0006] Some of the light passing through the analytical region will bescattered by particles. Detectors measure the intensity of forward andside scatter. In addition, the illumination beam will excitefluorescence from target particles in the flow stream that have beenlabeled with a fluorescent dye. Emitted fluorescence is collected by acollection lens and transmitted to detection optics. The detectionoptics separate the collected light (e.g. using filters and dichroicmirrors) into light at specific wavelengths. Light at specificwavelengths, or within specific wavelength ranges, are detected byindividual light detection devices (e.g. photomultiplier tubes). Thesignal from the various detectors is sent to a data processor and memoryto record and characterize detection events.

[0007] In addition to analysis of particles, flow cytometer systems mayalso be designed to sort particles. After leaving the optical analysisregion, the flow stream may be separated into droplets. One commonmethod of droplet generation is to vibrate the nozzle from which theflow stream emerges. This may be done by vibration of the nozzle alone,or vibration of the entire flow cell. The resultant separated dropletsadopt a spacing which is a function of the stream velocity and thevibration wavelength. Droplets containing the target of interest arecharged by a charging device such as a charging collar. The chargeddroplets are directed between two charged deflection plates, whichangularly deflect charged droplets. The deflected droplets are thencollected in containers positioned in the path of falling deflectedparticles.

[0008] Known flow cytometry similar to the type described above aredescribed, for example in U.S. Pat. Nos. 3,960,449; 4,347,935;4,667,830; 5,464,581; 5,483,469; 5,602,039; 5,643,796 and 5,700,692. Allreferences noted are hereby expressly incorporated by reference.Commercial flow cytometer products include FACSort™, FACSVantage™,FACSCount™, FACScan™, and FACSCalibur™ systems all manufactured by BDBiosciences, the assignee of the instant invention.

[0009] The described system presents a number of advantages for theanalysis of particles (e.g. cells), allowing rapid analysis and sorting.However a number of limitations to the system exist.

[0010] Alignment

[0011] The system requires precise alignment of various elements tofunction properly. The lasers must be precisely positioned to properlydirect light to the objective. To aid in this positioning, the laser orother illumination source is commonly mounted on an x-y-z stage,allowing three-dimensional positioning of the laser. The steeringmirrors for the laser beams must be precisely positioned to properlydirect the illumination beam to the objective. This generally requiresthat the mirrors be mounted to allow for angular adjustment. Theillumination lens system must be exactly positioned such that theillumination lens focuses the illumination light onto the target area.This lens is also generally mounted such that it can be repositionedalong the x-y-z axes.

[0012] The flow cell must be positioned such that the angle at which theillumination beam impinges the flow stream and the distance from theflow stream to the illumination lens does not change. Commonly the flowcell is mounted on a stage, which allows x-y-z positioning of the flowcell. In addition the stage holding the flow cell may also allow forangular repositioning of the flow cell (e.g. α and θ positioning). Thisangular adjustment is critical for sorting, which requires preciseprediction of the sort stream direction. In addition, the optics usedfor detection of scattered light and fluorescence also must be properlyaligned.

[0013] The stream in air jet must also be aligned, to ensure that thestream in air is directed in the intended direction. This alignment iseffected by angular rotation of the flow cell. This alignment isadditionally important if the optical interrogation of the stream takesplace in a stream-in-air. The alignment procedure for a stream in airsystem requires first locating the stream-in-air with respect to boththe illumination and the light collection optics and then focusing eachof these components on a location within the stream in air.

[0014] Alignment requires user time and considerable user expertise. Attimes it is difficult to determine which element requires adjustment.Set up of the instrument generally requires a diagnostic of alignmentwith elements realigned by repositioning as needed. This occurs at leastonce a day, more frequently if an element is replaced or removed.Realignment necessitates both instrument down time and user time andexpertise. The time required to perform the alignment procedure ishighly dependent on both the condition of the system and the skill ofthe operator. In addition, the need for constant realignment reduces therepeatability of system performance.

[0015] A few attempts have been made to address the problem of the needfor repeated alignment of some elements of a flow system. U.S. Pat. Nos.5,973,842 and 6,042,249 to Spangenberg disclose an optical illuminationassembly for use with an analytical instrument. This assembly mayinclude an illumination source (e.g. a laser), a spatial filter, a beamshaping aperture and a focus lens. All elements are illumination opticalelements, not the flow cell or light collection elements. Each componentis mounted on a plate, frame or mounting cylinder, which in turn aremounted on a platform. Each of the plates or frames is movable along twoaxes by micrometer adjustments using adjusters with opposing springplungers. Following an initial adjustment, the plates or frames aresecured into a fixed location using screws or other devices to fix theplates or frames into place. The adjusters or springs are removed oncethe frames or plates are secured. The focus lens would be mounted suchthat it would be moved along 3 axes (x-y-z movement) and subsequentlyalso be fixed into a location. This allows fixation of the lightgeneration and illumination optics. However, the cuvette would still beadjusted to be positioned at the focal spot of the illumination. Thiswould be required on a routine basis.

[0016] U.S. Pat. No. 4,660,971 discloses an illumination configurationin which a focus lens is in contact with a flow cell. A spring biasesthe lens against a housing, positioning the lens at a selected focallength from the flow cell. This maintains a relative axial positionbetween the lens and the flow cell.

[0017] These references, while providing a method in which some of theissues relating to the alignment of the illumination optics areaddressed, do not provide a method in which the flow cell and the lightcollection optics may also be fixed. Fixing all of these elementssignificantly further simplifies the alignment of the instrument.

[0018] Illumination Power

[0019] A number of different features in a common flow cytometer setupresult in loss of illumination intensity or loss of intensity ofcollected light. To compensate for these losses generally requiresincreased illumination power. This requirement for increased powerrequires expensive and bulky liquid-cooled lasers that providesufficient power to overcome losses and still allow sensitive targetdetection. These sources of loss include:

[0020] 1. Optical interrogation using a stream-in-air. The grosscylindrical geometry of a stream of liquid in air acts as a lens bothreflecting and refracting illumination light. This high index ofrefraction is more pronounced in smaller diameter streams. Thisrefraction makes illumination less efficient and distorts light scatter.To mitigate this effect of scatter distortion an obscuration bar ispositioned between the stream in air and the light scatter detector. Insome systems, this rectangular obscuration bar may be rotated to blockadditional amounts of light scatter across a greater area, blockingadditional light from narrow angles from reaching the scattered lightdetectors.

[0021] 2. Use of dichroic mirrors to combine illumination beams. Eachdichroic mirror is not able to perfectly reflect or transmit a lightbeam. As the beam is reflected or transmitted some light is lost. Thisloss ranges from 10-20% of beam power (5-10% if beam is reflected;10-15% loss for transmission through a dichroic mirror), more if thedichroic is not perfectly aligned. A laser beam that is reflected by asteering mirror through two dichroic mirrors to combine three beamscould lose 40% or more of the laser's power.

[0022] 3. Losses in collection of fluorescence. The amount of collectedfluorescence can be limited by the optical properties of the flow celland the collection lens. The geometry of the flow channel and the flowcell define the numerical aperture from which the system is able tocollect light. The transition from the flow cell to the collection lenscould allow refraction of light and loss of signal as the emittedfluorescence travels through the flow cell, into air, and then into thecollection lens. The high index of refraction during material transitionresults in the loss of collected light. In some systems this loss ismitigated by physical coupling of the flow cell to the collection lens.However, this coupling would be greatly simplified if the flow cell werein a fixed location.

[0023] Droplet Generation

[0024] Droplet generation has required vibration of some part of theflow cell, generally either the nozzle or the entire flow cell.Vibration of the entire flow cell can result in alignment difficulty aswell as additional light scatter created by the vibration. In addition,if the optical analysis is performed in a stream-in-air, the drop-driveperturbations cause undulations on the free surface of the stream. Thiscauses a constant alteration of the light paths into and out of the jetof liquid, making measurement of scatter and focusing of theillumination beam more difficult.

[0025] U.S. Pat. No. 6,133,044 provides one alternative to the vibrationmethod of droplet generation. This reference describes a device in whichan oscillator is included within the nozzle volume or otherwise isundirectionally coupled to the sheath fluid. The tapering of the nozzleamplifies the oscillations, which are transmitted as pressure wavesthrough the nozzle volume to the nozzle exit. This results in theformation of droplets. The nozzle is directionally isolated to avoidvibration of the entire flow cell or nozzle and limit the oscillationsto forming pressure waves in the flow stream.

[0026] Optics Positioning Limitations

[0027] Ideally, the flow cell would be materially joined to the lightcollection optics to prevent the loss of collected light. One of thegreatest losses of collected light occurs due to the transition betweendifferent materials that each have a different idex of refraction oflight. The light refraction between different materials (e.g. air andglass) may be significant and the resultant light refraction makes thecollection and measurement of scattered or fluorescent light difficult.This is mitigated by joining the flow cell to the light collection lens.However for the flow cell and the light collection lens to be coupled bya physical material would require that the two elements remain in afixed location.

[0028] In addition, the need to guard the flow cell from damage (e.g.scratching of surfaces through which light passes) presents anothermotivation for keeping the flow cell at a fixed location.

[0029] Flow Cell Positioning Limitations

[0030] Sorting flow cytometers generate a stream of droplets in air andsubsequently sort droplets containing target particles. The dropletstream is generated from a flow nozzle positioned at one end of a flowchannel. A large degree of uncertainty in the nature of the stream ofdroplets is a common result of the way in which the nozzle is located tothe flow channel. Most flow designs rely on the “self-aligning” tendencyof a female conical structure at the nozzle inlet, which mates with anedge on a cylindrical structure at the flowcell outlet (i.e. the outletof the flow channel). Typically an o-ring makes a seal between thenozzle conical structure and the flow channel cylindrical structure.

[0031] However, there are a few problems inherent with this approach.First, the o-ring has a compliance that aggravates the axial and angulartolerance stack-up associated with locating a conical surface about acircular arc. Second, the angular location of the nozzle about the axisof the flow cell is arbitrary. Third, the angular location of the o-ringabout the axis of the flow cell is arbitrary. The first noted problemmakes it difficult for a user to duplicate the mounting of the nozzle toa previous mounting configuration. The second and third noted problemsmake it impossible. Because the angular location of the nozzle and theo-ring are arbitrary, the nozzle is not formally constrained withrespect to the flow channel (or the cuvette) through which the flow cellextends.

[0032] U.S. Pat. Nos. 6,263,745 and 6,357,307 to Buchanan et al.disclose a nozzle for sorting flat samples. This nozzle seats in acylindrical recess in the flow cell. U.S. Pat. No. 6,133,044 discloses aremovable nozzle for use with a flow cytometer. The nozzle seats in acylindrical recess in the flow body and is held against a lip. Anannular nut secures the nozzle to the body of the tapering flow cell. Ano-ring positioned between the nut, the nozzle and the tapering flow cellprovides a means for ensuring the axial orientation of the nozzle.

[0033] Cell Sorting

[0034] Cell sorting requires precise coordination of event detection,droplet generation and droplet tagging. If these procedures become evenslightly out of coordination, the incorrect droplets could be chargedfor sorting or the system could fail to collect the desired particles orcells. For stream-in-air analysis and sorting, this process issimplified because the droplet stream is optically analyzed, dropletsare generated and droplets charged all in a stream in air. However, asnoted earlier, the stream-in-air sorting produces a decreased signalfrom cells or particles sorted and the circular stream of liquid cancause both illumination light and scattered light to be reflected orrefracted.

[0035] Sorting using a system in which analysis is done in a channelalso presents challenges. When the liquid moves from an analysis channeland subsequently through the nozzle the velocity of the particleschanges, as the liquid flow accelerates at the narrow nozzle. Thecoordination of flow must account for this change in flow rate.

[0036] U.S. Pat. No. 6,372,506 to Norton discloses an apparatus andmethod for determining drop delay. Drop delay is the time that elapsesbetween detection of a target at an analytical region to the time atwhich a sorting condition (e.g. a charging potential) is applied to thedroplet. As the droplets are formed they are analyzed to determinewhether the drop delay is correct. The droplets are analyzed todetermine if the target detected at an analytical region is within thedroplet to which the sorting condition is applied.

[0037] As fluid enters a channel, flow over a short distance can bemodeled as “slug flow”, all liquid moves as a single front. This wouldbe the case at the entrance of the neckdown region of the flow cell. Asliquid moves along the length of the channel, the viscosity of theliquid produces a parabolic velocity profile. The velocity of the liquidflowing through the cuvette channel tube is fastest along thelongitudinal axis of the tube. At the walls of the tube the fluid has novelocity. At any intermediate point between the walls and the center ofthe channel, the velocity of the fluid varies parabolically. Thislaminar flow results in a spreading of the distance between particles atdifferent distances from the tube center as the particles move throughthe stream. Particles in the exact center of the stream will move fasterthan the particles closer to the edges of the stream.

[0038] The laminar flow produces a spread of particles as the particlesmove through the channel. This can make sorting particles opticallyanalyzed in a cuvette channel more difficult. If the velocity of aparticle changes as the particle moves through the channel and opticalinterrogation occurs in the channel, the velocity of the particle at thepoint of optical interrogation and the velocity of the particle at thepoint of exiting the channel through a nozzle will be significantlydifferent. Since prediction of the position of the particle depends onknowing the velocity of the particle, sorting particles becomes muchmore difficult if the velocity of the particle changes.

[0039] It is an object of the invention to provide a flow cytometer thatrequires alignment less frequently, and most preferably only at aninitial instrument setup.

[0040] It is a further object that this system allows for efficientillumination and collection of light.

[0041] It is a further object that the losses of illumination light bereduced to allow for lower power lasers to be used for illumination.

[0042] It is a further object to provide a system that is easier to useand provides robust system performance.

SUMMARY OF THE INVENTION

[0043] The present objects of the invention are achieved through anumber of embodiments of the invention in which elements of a flow cellor flow cytometer system are designed for efficient light collection,efficient droplet production, and minimization of the need for usermanipulation of the system.

[0044] In one embodiment the invention includes a removable nozzle key,which fits into a registered location on a flow cell at the end of aflow channel. Clogs are an issue: The customer-removable nozzleaddresses this with no subsequent alignment required. The nozzle key maybe inserted into a registered location on the flow cell such that thenozzle is precisely positioned. The nozzle key may be removed, cleaned,refit into its precise location.

[0045] Removal of the nozzle allows the flow cell to be attached at afixed location on a system platform. If the flow cell position is fixed,other optics that must be positioned relative to the flow cell may alsobe fixed. This allows the illumination optics, the fluorescent lightcollection optics and the scattered light detection optics to also be ina fixed location.

[0046] The fixed illumination optics may include fixed optics fortransfer of the illumination beams into the system and fixed optics forbeam shaping and orientation. The optics for bringing the illuminationlight into the system could use optical fibers coupled into the systemat fixed location mounts. The optics for shaping and orienting the beamscould be refractive optics, which are less alignment sensitive than themirrors used in prior systems for beam redirection and shaping.

[0047] The light collection optics may also be fixed. If the flow cellis fixed and the light collection optics is fixed, the flow cell may bematerially coupled to the light collection lens, as by gel coupling.This lowers losses to refraction.

[0048] The design of the present system's elements aids in efficientlight collection. In one embodiment, the cuvette containing the flowchannel has sidewalls extending on three sides of the cuvette below theplane containing the opening of the flow channel. Light emission fromthe flow channel may pass into the sidewalls and subsequently into thelight collection optics. This allows for light collection from a greaternumerical aperture than is seen in prior systems. This design alsoallows the optical analysis to take place quite near the bottom of theflow channel. This makes determination of the drop delay (needed forcharging generated droplets for subsequent sorting) simpler. Inaddition, there is less variability between particles of differentvelocities. Many of these described features are independent embodimentsof the present invention.

[0049] In another embodiment of the invention, a flow cell for a sortingflow cytometer is provided in which a removable nozzle is inserted intoa registered position in which it is held at a fixed location inrelation to the rest of the flow cell. This fixed position prevents thenozzle from either three-dimensional or rotational movement.

[0050] Flow cells include a sample delivery tube, at least one sheathflow port, and a channel for optical analysis. This channel may be partof a flow cell body, but preferably is a cuvette joined to the flow cellbody. When the cuvette is joined to the flow cell body, the sheath flowand sample stream flows into the cuvette.

[0051] Flow cells for a sorting flow cytometer also include a dropletgenerator. The droplet generator would ether vibrate an element on theflow cell, such as the nozzle, cuvette or flow cell body, or wouldintroduce a oscillating pressure wave within the flow cell body.

[0052] The removable nozzle is held on a substrate, such as a card orinsertable key, which is fit into a registered position in which thesubstrate is registered against hard surfaces, allowing the substrate tobe removed and replaced into a precise position.

[0053] In another embodiment, the sorting flow cell includes anoscillating droplet generator that transmits a pressure wave to thesheath flow fluid flowing through the flow cell. In this embodiment,droplets may be generated without a device for vibrating the flow cell,the cuvette, or the nozzle. A number of noted features, including theregistered nozzle, may be included with this embodiment.

[0054] In another embodiment, the sorting flow cytometer flow cellincludes a flow channel of rectangular cross-sectional dimensions. Theshorter side of the channel would face the optical path of theillumination light directed by the illumination optics. The longer sideof the channel would face the light collection optics. Thisconfiguration has a high numerical aperture for collection of emittedlight. The channel may extend through a cuvette. The cuvette may havesidewalls that extend below the area of the nozzle, allowing a widerangle to collect emitted light. This configuration also allows anenhanced numerical aperture of collected light compared to systemslacking the sidewalls. Again, a number of noted features, including theregistered nozzle, may be included with this embodiment.

[0055] In another embodiment, a flow cell component includes a flow cellfor a sorting flow cytometer and light collection lens. The flow cell isjoined to the light collection lens by a light transmissive material. Inthis manner there is no transition into air as light moves from the flowcell to the light collection lens. This reduces the loss due to thechange in index of refraction as light moves from the flow cell, intoair, and then into the collection lens. In prior non-sorting flowsystems, cuvettes could be joined to the collection lens. However, insorting systems, the need to clear the nozzle and vibrate the flow cellmade joining the collection lens and the flow cell inadvisable. In thepresent invention, these limitations have been overcome, and theadvantages of joining the flow cell to the collection lens are achieved.

[0056] In another embodiment, the flow cell for a sorting flow cytometerincludes a nozzle held at a hard registered location that is off centerfrom the longitudinal center of the channel, this is possible becausethe nozzle may be removed and reinserted into a precise location. Thisprovides favorable conditions for the formation of droplets and themerger of satellite droplets into parent droplets.

[0057] Each of these embodiments represent a component which may beindependently produced. Alternatively, each component (flow cell or flowcell with optics component) may be part of a flow cytometer system. Sucha system would include light collection optics and illumination lightdirection optics. The system could also include input optics that wouldallow illumination light sources to be coupled to the system.Alternatively, the system could be produced with the illumination opticsas an already fixed part of the system. In addition, the features ofeach embodiment may be incorporated with the features of otherembodiments if desired.

BRIEF DESCRIPTION OF THE DRAWINGS

[0058]FIG. 1 is a perspective view of a flow cytometer systemincorporating features of the present invention.

[0059]FIG. 2 is a side view of the system of claim 1.

[0060]FIG. 3 is a frontal cross section of a flow cell, nozzle andnozzle support platform.

[0061]FIG. 4 is a side cross section of the device of FIG. 3.

[0062]FIG. 5 is a detail of the nozzle and flow cell shown in FIG. 3.

[0063]FIG. 6 is a detail of the nozzle shown in FIG. 4.

[0064]FIG. 7 is a detail of the nozzle from FIG. 5.

[0065]FIG. 8 is a cross section detail illustrating positioning of thenozzle in the flow channel.

[0066]FIG. 9 is a top view of the nozzle card.

[0067]FIG. 10 is an exploded view of the nozzle card.

[0068]FIG. 11 is a frontal detail view of the light collection optics ofFIG. 2.

[0069]FIG. 12 is a top view of the nozzle card, flow cell, andfluorescence collection lens.

[0070]FIG. 13 is a side cross sectional view of the devices shown inFIG. 12.

[0071]FIG. 14 is a perspective view of the devices shown in FIG. 12.

[0072]FIG. 15A is a view of droplet formation.

[0073]FIG. 15B is a view of droplet formation using the technology ofthe present invention.

DETAILED DESCRIPTION OF THE INVENTION

[0074] In the present invention, significant advantage is derived from aconfiguration in which a number of the optical elements may be fixedwith respect to the flow cell. This advantage arises from the extent ofdirectional stability afforded by the nozzle, which the user may removeand replace and which is self-aligning. The nozzle is insertable in theflow cuvette at a location where the nozzle is registered in place. Thisregistration allows the nozzle to be inserted and positioned such thatthe nozzle is constrained both as to translation and rotation. Becauseonly the nozzle is movable, the flow cell may be fixed, and does notneed to be positioned on a stage that may be angularly or directionallyrepositioned. As a result, no removal or replacement of the flow cell isrequired and the user will not have to adjust or realign the flow cellassembly to align the stream of droplets with a required direction forsorting.

[0075] Because the flow cell and flow channel never need to be moved,the other optical elements that must be focused or positioned relativeto the flow cell now may be fixed as well. This fixation may be locationof the flow cell on one fixed plate and the location of other opticalelements on one or more additional fixed plates, with each plate in adefined positional relation to any other plate. Alternatively, the flowcell could be materially linked to other optical elements, such as byphysical joining of the flow cell with the collection optics. Materiallyjoining the flow cell to the light collection optics allows reduction ofthe index of refraction between material transitions and allows moreefficient collection of light.

[0076] The present configuration minimizes tolerance stack-up. Only thefabrication tolerances of two mating elements, the nozzle and thereceiving flow cell body can contribute to the stack-up. These are theonly two elements that would be moved in relation to the other. Anintermediate locating element between the nozzle and the cuvette would,at least, double the tolerance stack-up and adversely affect streamstability.

[0077] With reference to FIGS. 1, 2 and 11 the system allowing fixedposition mounting of the elements is shown in a perspective view. Thelasers (not shown) produce illumination light, which is directed throughan optical fiber, linked to the system by mounts. The first, second andthird optical fiber mounts 11, 12, and 13 respectively each receive anoptical fiber bringing illumination light from one laser. Optical fibermounts are mounted on plate 19 that is secured to platform 20 by braces22. Braces 22 ensure that plate 19 will be maintained in a fixedposition. Light from optical fibers coupled to mounts 11, 12, and 13 isdirected through illumination refracting optics.

[0078] A series of prisms are used to combine the illumination beamsinto illumination light having specific properties. At the point ofillumination, it is preferred that the illumination beams be elliptical,concentrating the illumination energy at the central location of thecore of the flow stream. As the light is directed through prisms 23, 24,25 and 26 the illumination beams are differentially refracted by theprism such that the illumination beams are redirected and aligned at theillumination location within a flow cell. Prisms 23, 24, 25 and 26 aremounted on plate 21, which is secured to platform 20 by braces 27. Themounts for both the laser couplings and the prisms may be adjusted onceat the setup of the system, positioning each element in a fixedlocation. This may be performed by either adjusting the mounts for eachelement, or repositioning the plate on which elements are mounted sothat a number of elements are moved together. The prisms are lesssensitive to angular misalignment and are more thermally stable. Both ofthese features aid in allowing set position of this element.

[0079] The illumination beams pass through the illumination beamcombining optics and through illumination lens 30 held in illuminationlens mount 31. Illumination lens mount 31 is positioned on arm 60secured to face 61 on lens positioning stage 33. Micrometer 35 extendsto arm 60 to allow movement of lens mount 31 along the z-axis. Plate 61is movable by micrometer 34 to allow movement of the illumination lensmount along the y-axis. Micrometer 36 is mounted through lenspositioning block 33 to allow movement of lens mount 31 along thex-axis. In combination, positioning micrometers 34, 35, 36 allow lensmount 31 to be repositioned in the x, y, and z directions. Lens mount 31is mounted on block 33, which is mounted on platform 20. Plates 19 and21 and block 33 are each at a separate location on platform 20. Each ofthese elements may be separately adjusted initially at installation foralignment.

[0080] The focused illumination beams pass through the illumination lensheld in lens mount 31 and through the optical analysis region of theflow channel in flow cell 41. As particles pass through the flow channeland cross the illumination light beams, light will be scattered andfluorescence will be excited. Scattered light will be detected byforward light scatter detector 43. Emitted fluorescence as well as largeangle scattered light will be collected by emission collection optics.Flow cell 41 and forward light scatter detector 43 are mounted on plate39. Plate 39 is held on platform 20 by brace 29. Platform 20 issupported on feet 46. Isolation mounts 45 allow mounting of platform 20.

[0081] In the system of FIG. 1, the optics for bringing in light,redirecting the beams into the desired illumination orientation,collection of the scattered and emitted fluorescence are all held in afixed position. In addition, the flow cell is also held in a fixedposition. These elements are each mounted on a plate and need to bealigned initially when the instrument is installed. Routine realignmentof these elements will not be required. The illumination lens could beroutinely realigned. However, alignment of a single element greatlysimplifies the time and difficulty of alignment.

[0082] It should be realized that a number of the elements of thissystem have independent utility. For example, simply having a fixedlocation flow cell and light collection optics allows joining of theflow cell to the collection lens to more efficiently collectillumination light. Fixing the mount location of the beam redirectionoptics also saves user time and provides a method of combiningillumination beams into a single illumination beam without using mirrors(and the attendant loss of light characteristic of mirrors).

[0083] The term “fixed location” as used herein refers to an elementwhich does not have a means for user adjustment. This element at a fixedlocation would be aligned initially (generally at instrument set up) andnot require further alignment.

[0084] “Illumination input optics” refers to optical elements that allowintroduction of light to a system (e.g. optical fiber mounts).

[0085] “Illumination beam directing optics” are optical elements thatredirect and reshape the illumination beams (e.g. serial prisms).Illumination beam directing optics may or may not include a focus lens.

[0086] “Light collection optics” refers to optical elements disposed tocollect emitted or scattered light from the flow channel. In flowsystems such optics generally include a fluorescence and wide anglescatter collection lens and a forward scatter detector.

[0087] The ability to have a fixed position illumination input optics,illumination beam directing optics, flow cell, and light collectionoptics depends on two factors. The first is the ability to direct lightwith elements that will not routinely go out of alignment. The second isthe ability to fix the location of the flow cell, allowing the distancebetween the flow channel, the illumination optics and collection opticsto remain constant. This allows the illumination optics and thecollection optics to also be located at fixed locations. It furtherallows the flow cell to be joined to the light collection optics in away that minimizes loss of the collected light.

[0088] With respect to FIGS. 3 and 4, the flow cell 41 is shown in frontand side cross section respectively. The sheath flow tubes are not shownin these views. Generally sample delivery tube would be positioned suchthat the sample is introduced just before the neckdown region (theregion adjacent to the beginning of the flow channel) as shown in FIG.3. The two sheath flow delivery tubes provide sheath flow in an even,pulse free flow.

[0089] In FIG. 3, the flow cell 41 is shown comprised of flow cell body230, cuvette 210, nozzle key 214, and flow cell base plate 220. Flowcell body 230, cuvette 210 and flow cell base plate 220 are joinedtogether to form a single unit that may be secured in a fixed locationby bolts extending through holes 209 onto a flow cytometer instrument(e.g. by bolting the flow cell onto a fixed position plate). The nozzlekey 214 is not fixed and may be inserted into a location such that thenozzle is at the end of the flow cannel. The nozzle key could besubsequently removed, cleaned (e.g. sonicated) and reinserted. Inaddition, the fitting 233 containing the sample input tube also might beperiodically removed and reattached. This allows the remaining portionsof the flow cell to be in a fixed location within a flow cytometer.

[0090] The upper portion of the flow cell is the flow cell body 230,which receives both the sheath flow tubes and the sample delivery tube202. The sheath flow liquid is delivered in tubes joined to fluid inputbody though ports 206, 208. The flow is delivered such that the sheathfluid surrounds a core of the sample stream as liquid passes through theflow channel 212. The sheath fluid carries the core stream through aconverging channel in flow cell body 230 and into the flow channel 212in cuvette 210.

[0091] The flow cell body 230 has an open top end through which thesample tube and oscillator are introduced. Inserted through the open topend is sample tube inlet fitting 233 and transducer plunger 232. Plunger232 is retained on boss 202 on flow cell body 230. A tube (not shown)held by fitting 233 introduces a sample liquid through a passage infitting 233. This passage is joined to sample delivery tube 202 suchthat liquid flows through the passage, into sample delivery tube 202 andinto a passage within the flow cell body. The sample delivery tube 202terminates at an open end proximate to the flow channel 212 that extendsthrough cuvette 210. At this location the sample flowing through sampledelivery tube 202 is surrounded by sheath flow fluid, forming the sampleinto a core in the flow stream as the stream moves through flow channel212.

[0092] The flow stream flows through flow channel 212 in cuvette 210 andexits at nozzle 216. The length of flow channel H1 is sufficiently longto ensure fully developed flow in the optical analysis region H2 underall operating conditions. In the illustrated system a length of 8-15 mmis sufficient for a fully developed flow. Sidewalls 213 extend aboutthree sides of nozzle key 214, allowing registration of the nozzle key214 in place. At the point of exit, the sample stream flow velocityincreases as the sample exits the narrower nozzle opening.

[0093] Nozzle 216 is mounted on nozzle key 214, positioned at aregistered location at the end of cuvette 210. The bottom side of nozzlekey 214 rests on flow cell base platform 222 on flow cell base plate220. H1 indicates a height of the cuvette between the flow cell body 230and the nozzle key 214. This is the location where illumination beamsare directed through flow channel 212. Close tolerances between thenozzle and the registration features insure that the direction of thestream does not change after a nozzle has been removed and replaced bythe user.

[0094]FIG. 5 shows a detail of the nozzle key 214 and cuvette 210.Nozzle key 214 has a nozzle key card 213 affixed to the top surface ofthe nozzle key 214. The nozzle 216 is positioned on nozzle key card 213on the nozzle key 214 such that the nozzle 216 is positioned at aselected location in the cross section of flow channel 212 when nozzlekey 214 is inserted into cuvette 210. When nozzle key is inserted intothe registered position, nozzle key 214 is held between flow cell baseplatform 222 and the top surface of cuvette. The stream generated byflow through nozzle 216 flows into passage 218.

[0095] The detail of the nozzle is shown in FIG. 7. Cuvette 210 is shownhaving a flow channel 212. At a terminus of flow channel 212 nozzle 216is positioned. Nozzle 216 is shaped like a truncated funnel, producing amore stable flow stream. On nozzle key card 273, an annular groove 261holds an o-ring 260. O-ring 260 seals nozzle key card 273 to cuvette 210when nozzle key 214 is inserted in its registered position.

[0096] With reference to FIG. 3, the stream in air which flows fromnozzle 216 then passes through passage 218. The sample could becollected here or the stream could be separated into droplets, allowingsubsequent charging and sorting of droplets. To generate droplets, adrop drive piston 240 may be used. Signals for the power and operationof the drop drive piston 240 may be sent through transducer electricalterminal 235. The electronic signal is sent to drop drive piezo element234 held in transducer plunger 232. Drop drive piezo element 234oscillates drop drive piston 240, sending oscillating pressure wavesthrough the incompressible sheath flow fluid.

[0097] Previous systems have used vibration (as from a piezoelectriccrystal) of the nozzle cuvette, or flow cell to generate droplets. Thedroplets generated are separated by the wavelength of the vibration.This allows division of the flow into individual droplets for sorting.However, the vibration of either the nozzle or the entire flow cellcould have negative effects on the consistency of illumination and lightcollection if the vibration causes the relative distance from the flowchannel and the illumination focus or light collection focus to change.This effect is more pronounced in stream-in-air optical interrogation.In the present invention, droplet generation originates from adisplacement type oscillator near the source of the sheath flow. It hasbeen found that the pressure waves are transmitted through the largelyincompressible flow fluid and effectively generate the desired droplets.

[0098] With respect to FIG. 4, the side cross-section shows detail ofthe key nozzle 214 as it is secured in place. In the side view, key 214is shown having nozzle key grip 270. Nozzle key 214 has a passage 218defined by surface 254 and surface 256. Nozzle key plunger 281 biasesthe nozzle against the sidewall of the cuvette, holding the nozzle in aregistered position. The detail of the nozzle section shown in FIG. 6shows the insertion of the nozzle between cuvette 210 and flow cell baseplatform 222, holding nozzle key 214 in a position such that nozzle 216is registered against the terminus of channel 212 extending throughcuvette 210, by the cell base platform 222. Nozzle key plunger 281provides a biasing pressure to retain nozzle key 214 in position.Shoulder 311 on nozzle key 214 is appressed against a surface of cuvette210 when the nozzle key is fully inserted. This positions the nozzle ata registered position. The nozzle is prevented from being inserted toofar, preventing damage to back wall 254.

[0099] The details of the nozzle key are shown in FIGS. 9 and 10. InFIG. 9, nozzle key 214 is shown with nozzle 216 positioned on nozzle keycard 273. Label 271 affixed to the bottom of the card allowsidentification of the specific nozzle card used. Nozzle key plunger 281provides a biasing force of the nozzle key 214 against the side walls ofcuvette 210. Nozzle key grip 270 allows a user to grip nozzle key 214and remove it from cuvette 210. In this way if the nozzle were to becomeclogged, the nozzle could be simply removed, cleaned (e.g. sonicated)and replaced.

[0100] In FIG. 10, the nozzle key is shown in exploded view. The nozzlekey plunger 281 is inserted through the nozzle key 214. Spring 282,retained within nozzle key 214 by nozzle key spring plug 283, provides abiasing force on plunger 281. Plug 283 is retained on plug retainer 284.

[0101] Nozzle key card is affixed on the top of nozzle 214. O-ring 260fits into groove 261 to provide a sealing force of the key card 273 tothe cuvette when nozzle key 214 is inserted into the cuvette.

[0102] The use of a cuvette for the optical analysis of the streamallows for a lower excitation power requirements and greater efficiencyof the collection optics. As opposed to analysis in a stream in air, thecuvette presents a stationary target with a flat interface for theincoming laser light from the illumination optics. Therefore, less lightis lost to reflection and refraction. Because less light is lost, lowerlaser power is required. These features also make the collection oflight more efficient. Less light is lost due to refraction of light fromthe stream to the light collection optics. In addition, the materialtransition from the stream to the collection optics can avoid thetransition from liquid to air, with the attendant high index ofrefraction eliminated.

[0103] As noted in respect to FIGS. 1 and 2, the use of the nozzle thatmay be inserted into a registered position allows fixing the position ofthe flow cell, illumination optics and the light collection optics. Oneadvantage to this configuration is the elimination of wear and tear onthe flow cell. When the flow cell is removed, it is possible that thesurfaces through which light pass on the flow cell could becomescratched or marred such that light collection or transmission to orfrom the illumination channel is altered. This is mitigated by fixingthe flow cell in place and not requiring the flow cell to be moved ormanipulated.

[0104] As noted, each material through which light passes will have acharacteristic index of refraction. Light will be refracted when itpasses from a medium having a first index of refraction to a mediumhaving a second index of refraction. A major problem encountered inprior systems that optically analyze in a stream in air is the highindex of refraction between the stream of liquid and air. This, coupledwith illumination light losses due to the gross cylindrical nature ofthe stream in air, requires higher excitation power than is required ina cuvette system.

[0105] In a cuvette system, losses to refraction also occur in thetransition from the cuvette to air material transition as emittedfluorescence moves from the cuvette, into air and subsequently into thecollection lens. Fluorescence excited in a liquid moving through theflow channel is collected by a collection lens on one side of the flowchannel. If the flow cell is fixed in location, the light collectionoptics may be physically joined to the cuvette. This reduces therefraction at the material transition from the cuvette to the collectionlens.

[0106] With reference to FIGS. 12 and 14, the cuvette 210 is joined tothe emission collection lens in housing 50. The nozzle key 214 isinserted into position such that the nozzle is positioned at the end ofthe flow channel. This is shown in FIG. 13. In this cross sectionalview, the flow channel 212 is shown extending through the cuvette 210.The cuvette 210 is linked to an initial optical element 52 by a gel 290.Light is collected by lenses in housing chamber 51. One such collectionlens is disclosed in U.S. patent application Ser. No. 09/934,741entitled “Flow Cytometry Lens Systems”.

[0107] Nozzle key 214 is inserted and registered against cuvette 210. Inone direction this registration is effected by biasing the key by nozzleplunger 281, holding the nozzle card in position laterally.

[0108] The sidewalls 213 of the cuvette 210 extend below the exit planeof the cuvette (i.e. the plane containing the exit of the end of theflow channel). This allows for a larger numerical aperture for thecollection of emitted fluorescence and for forward scatter. In additionthe lower sidewalls 213 permit a lower entrance point for the laserbeams, enabling the closest possible location of the opticalinterrogation region to the nozzle. In one embodiment the opticalinterrogation is 700 nm from the nozzle opening. This configurationensures minimization of the delay time and least time delay errorbetween the lowest laser illumination of the stream (target detection)and the droplet charging.

[0109] In prior flow cytometers in which optical analysis of a sampleoccurs in a cuvette channel, the cuvette would be of a block shape andthe sidewalls would terminate at a bottom surface of the cuvette. Inthis configuration the illumination must occur a significant distancefrom the bottom of the cuvette, in order for efficient light collectionof emitted fluorescence. If illumination occurs too close to the bottomof the cuvette, much of the potentially collectable fluorescent lightwill be lost from the bottom of the cuvette, which would refract thelight away from the collection lens. To avoid this problem, the lightcollection would occur a significant distance from the bottom of thecuvette and thus a significant distance from the end of the flowchannel. This may be acceptable for non-sorting applications, but forsorting applications the separation distance of the detection of targetsand the nozzle is critical for determining drop delay and properlycharging and deflecting droplets of interest. It is also criticaltowards avoiding time delay errors, which reduce sorting performance.

[0110] In addition to the lower sidewalls, the geometry of the channelalso allows for more efficient illumination and light collection. In thepresent illustration, a rectangular cross sectional channel is used. Theshorter side of the channel faces the illumination light and the longerside of the channel faces the collection lens. This allows collectionfrom the area of the longer side of the channel. This presents a highernumerical aperture for collection. In FIG. 8 this is indicated by angleα. Collection from a higher numerical aperture allows more efficientcollection of emitted light and greater sensitivity. This greatersensitivity enables use of lower power lasers. In addition, this wideviewing window allows keeping the cross-sectional area relatively small.This reduces the volumetric consumption of sheath flow required for thesystem.

[0111] The system shown in FIGS. 1 and 2 would be contained in a housing(not shown). This housing would prevent light from the area surroundingthe system from entering the system.

[0112] Sorting droplets requires precise coordination of the detectionof a target of interest, encapsulation of the target into a dropletduring droplet formation, charging the droplet and sorting the dropletby passing the charged droplet containing the target of interest betweentwo charged deflection plates. The flow of fluid into the flow cell iskept pulse free so that the perturbations of the fluid are minimized.This allows the general condition of directional stream in airstability.

[0113] It is desirable to have the flow stream break up into droplets ina predictable manner. In a sorting flow cytometer, the drop drive causesa leading order effect in which the flow stream-in-air, after flowingfrom the nozzle, breaks into a train of large droplets having acharacteristic diameter of the same order of magnitude as the jetdiameter, as shown in FIG. 15a. Due to the nonlinearity of the fluiddynamics characteristic of flow cytometry, smaller droplets 306 atypically form between the larger “parent” droplets 304 a. The smallerdroplets are referred to as “satellite droplets”. It is advantageous tohave a stream condition in which no satellites form, or in which thesatellite droplets that do form quickly merge into the parent droplets.The satellite droplets are significantly smaller, and hence have lowermasses, than the parent droplets.

[0114] During particle sorting, sorting is accomplished by selectivelycharging droplets. The droplets then pass through an electric field thatdeflects the path of the charged droplets so that the charged dropletsare deflected from the rest of the droplet stream. The deflecteddroplets are deflected into a separate collection container for lateruse or analysis. The required magnitudes of both the droplet chargingand electric field potentials of the charging plates are selected toprovide the needed deflection of the parent droplets. The smallersatellite droplets that are deflected by the charged deflection platesmay be so light that the particles are directed out of the flow streamand onto the charging plates. The resulting wetting of the chargingplates may adversely affect system performance and require interruptionof the use of the system to dry and clean the deflection plates. Inaddition, the deflection of satellite droplets could present biohazardrisks, especially if the satellite droplets form aerosol droplets thatremain suspended in the air.

[0115] In the prior systems, favorable satellite conditions wereachieved through trial and error. A user could make ad hoc adjustmentsto the drop-drive amplitude, drop-drive frequency, and sheath pressureuntil a favorable satellite droplet conditions are achieved (i.e.satellites quickly merged with parent droplets). This is largelyguesswork, requiring a knowledgeable user and some time. Optical systemsthat monitor droplet formation are required to determine that thesatellite droplets are merging with the parent droplets.

[0116] Theoretically, a perfectly symmetric jet excited near itsspontaneous drop frequency will break into a droplet chain whosesatellite droplets never merge with the parent droplets. A portion ofthe drop drive energy cascades into a secondary satellite dropletformation harmonic, in phase with the fundamental droplet formationfrequency. Some experiments have shown that one method to control thesatellite formation is to add a phased, higher-harmonic component to thedrop drive vibration or pulsation to alter or cancel satellitedevelopment. (see Chaudhary, K C, and Redekopp, L G, “The nonlinearCapillary Instability of a Liquid Jet. Parts 1-3” J. Fluid Mech., Vol.96 (1980a-c)).

[0117] Location of the nozzle in a precise location in relation to theflow channel allows creation of a repeatable and favorable satellitedroplet merging conditions. One embodiment of the present invention usesthe nozzle location to ensure more optimal satellite droplet merging.With reference to FIG. 8, a detail of the cross section of the cuvette210 shows the flow channel 212 with a circle indicating the nozzleopening 294. The center 292 of nozzle opening 294 is positioned offcenter from the cross sectional center of flow channel 212. A smalllateral adjustment of the centering of the nozzle in the flow channelprovides a more favorable condition for merger of the satellite dropletswith the parent droplets.

[0118] In one embodiment of the invention the nozzle is purposefullymisaligned with respect to the center of the flow channel. Thismisalignment may be achieved by machining 0.001 inch from the nozzleregistration feature that locates the nozzle in the long dimension ofthe cuvette channel (e.g. the sides of the nozzle card). The presentsystem allows precise location of an insertable nozzle card. The abilityto precisely and repeatably locate the nozzle allows design of thenozzle card such that the nozzle is off from the flow channel center.

[0119] The nozzle location is fully constrained by hard features on thenozzle that register directly against the cuvette (or flow cell) suchthat the nozzle orientation is always fixed in three dimensions withrespect to the exit of the cuvette channel. The registration of thenozzle in this manner minimizes tolerance stack up. The limiting factorsof this approach are the manufacturing tolerances associated withmanufacturing the nozzle and the cuvette. State of the art manufacturingprocedures allow nozzle location to +/−0.0012″ (+/−30.48 μm) in theplane of the channel. Given these manufacturing tolerances,nozzle-to-nozzle stream performance has proven to be consistent. A givennozzle will always register against a given cuvette in the same manner,ensuring a consistent stream direction and droplet formation pattern.

[0120] As noted, the theoretical model of droplet formation indicatesthat an axisymetric flow stream excited by a fundamental frequency inthe range of the spontaneous droplet frequency will break up into adroplet chain in which the satellite droplets never merge with theparent droplets. This implies that deviation from perfect axisymetrycould allow for more optimal satellite droplet merging conditions.

[0121] In the present illustration, the nozzle is adjusted laterally inthe long dimension. This is generally preferred, as it produces thesmallest variation in the path for particles within the flow stream.This minimizes the difficulty in timing the delay between detection inthe channel and charging and sorting droplets after the droplets havepassed through the nozzle. It is also possible to have the displacementin the shorter dimension or displace the centering in both the long andthe sort dimensions (i.e. displace on a diagonal from the crosssectional center of the stream).

[0122] With reference to FIG. 15a, an image of the stream shows dropletformation in a system in which the center of the nozzle opening isclosely aligned with the center of the flow channel. At the top, thestream has begun to break into individual droplets. Height A marks thedistance from the formation of the first droplet broken from the flowstream to the area where the satellite droplets have combined into theparent droplets. It would be preferred that droplet deflection not occurbefore this location. Parent droplet 304 a and satellite droplet 306 aare identified close to the location of droplet formation at the top ofheight A. A merging parent and satellite droplet 305 a are identifiedclose to the bottom of height A. Eight or more droplet wavelengths arerequired before the parent droplet and the satellite droplets havemerged.

[0123] With reference to FIG. 15b, an image of the droplet formationpattern in which the nozzle has been misaligned is shown. This is themisalignment shown in FIG. 8, in which a rectangular flow channel isused. The dimensions of the flow channel in this embodiment is 250 um by160 um. The nozzle is deliberately shifted 25.4 um off of the centerlineof the cuvette in the long axis of the cuvette. In FIG. 15b, height B isa height from the location of a droplet formation to a location wherethe droplets do not show any satellite droplets. Satellite droplet 306b, shown just after droplet break-off point, and parent droplet 304 bare shown. A merging parent droplet with a satellite droplet 305 b isjust below. In three droplet wavelengths the parent droplets have mergedwith the satellite droplets.

[0124] It will be readily appreciated by a person of ordinary skill inthe art that a number of modifications to the present invention arepossible. The nozzle opening, as presently illustrated, is a truncatedtapered cone. A card is affixed to a nozzle key such that the nozzle isin a precisely registered location when the card is inserted into theflow cell. The nozzle may take many shapes and geometries. For example,an elongate nozzle opening may be preferable in some applications to around opening. The nozzle may be a lengthened truncated cone, extendingdownstream from the flow stream direction. In this way the transitionfrom the width of the flow channel to the nozzle may be made lessabrupt, and could be a continuous graded tapering to the nozzle opening.In addition, the substrate on which the nozzle opening is formed may usea number of different designs.

[0125] A number of the elements of the present invention could be usedindependently or in a number of different combinations. The fixed flowcell with nozzle key may be adapted into present systems in which theillumination optics and light detection optics may be aligned.Alternatively, the fixed flow cell may be used with either fixedlocation light collection optics (e.g. fluorescent light collector andlight scatter collectors) or a fixed location illumination optics. Otheroptical elements within such systems would still require routineadjustment. Such systems would retain the advantage of having a fixedposition flow cell that does not require to be removed or adjusted. Insome embodiments, the cuvette may not be physically coupled to the lightcollection optics. Such embodiments could have all of the attendantoptics (illumination optics, light collection optics, and scatterdetection optics) in a fixed location or have some or all of theseelements mounted on an adjusting mounts.

[0126] The droplet drive may be generated by an oscillator within theflow cell, allowing transfer of the oscillating pulse to the largelyincompressible fluid. Alternatively, the droplet drive may be generatedby more conventional means, such as vibrating the nozzle, cuvette orvibrating the entire flowcell assembly. Additionally, the droplet drivemay be generated by acoustic vibration of the stream in air.

[0127] In the illustrated embodiment, the flow cell body is joined to arectangular cuvette and the nozzle is inserted at the terminus of thecuvette. The term “cuvette” in various embodiments, is the flow cellsection through which the channel extends. This may be a separatecomponent joined to a flow cell body. Alternatively, the cuvette may bepart of the flow cell body, which may be manufactured as a single part.

[0128] In the preferred embodiment, the nozzle is on a substrate that isinserted into a fixed position where it is registered against surfacesto hold the nozzle in a three-dimensional position such that the nozzlecannot angularly rotate. This allows the cuvette and flow to be in afixed position. Because the channel is in a fixed position, theillumination optics and light collection optics may also be in a fixedposition. In addition, the cuvette may be physically joined to thecollection lens. It is also foreseen that a flow cell could be made inwhich the nozzle is a fixed part of the cuvette and the cuvette andnozzle are removed and inserted together. Precision guides could be usedto precisely position the cuvette at the required location needed foralignment with illumination optics. The cuvette could be removed,sonicated or otherwise treated to clear a clog in the nozzle or flowchannel, and replaced into a precise position. Because the dropletgenerator is in the flow cell body, the cuvette and nozzle could beremoved and reinserted without having to reconnect to a vibrationgenerator.

[0129] The invention was illustrated in a system in which multiplelasers are directed into the system using optical fibers. The beams areredirected and shaped using refractive optics. It is envisioned that asingle laser or any number of lasers may be used. The lasers could bepositioned on the platform and held in a fixed position (e.g. usingdiode lasers). It is also possible to employ non-laser light sourcessuch as arc lamps. In place of the refractive optics, the conventionaluse of steering mirrors and dichroic mirrors could be used to direct andshape the illumination beams. In addition, spatial filters, long orshort pass filters, apertures or other optics may be employed to blockstray light and reduce transmission of undesired wavelengths. Systemsemploying conventional optics are disclosed in the references citedherein, which are collectively incorporated by reference.

[0130] The flow channel in the present illustration is rectangular. Inother embodiments, a round flow channel or other geometries areenvisioned. In the illustrated embodiment, nozzle is on a removable key.However, the nozzle could also be precision aligned and affixed (bysonic welding, adhesive attachment, etc.) to the end of the flowchannel. Such a system would require some means of back-flushing thenozzle to clear clogs.

[0131] The illustrated system discloses the use of the flow cell in asorting flow cytometer. While the present invention provides numerousadvantages when employed in a sorting cytometer, it is also consideredthat disclosed technology could be used in non-sorting analyticalcytometers.

[0132] The interior of the flow cell could be coated to prevent adhesionof cells, cell fragments or other compounds. Such a treatment would beselected to not be affected by the flow fluid. The flow cell is notremoved but may be selectively flushed. The sheath flow system may bedesigned to allow for system flushing. If fluid is introduced through afirst sheath flow port and removed through a second port, flowconditions would direct the flow in a vortex through the flow cellinterior, washing all elements.

[0133] Because the nozzle is smaller than the flow channel, any clog islikely to occur in the nozzle, which may be removed and cleaned. If thechannel clogs, the nozzle may be removed and the channel cleared withback pressure.

[0134] The present invention allows development of a system in which theuser does not have to perform any routine optical alignment procedure.The user could remove and replace the nozzle without further alignmentof the stream direction or optics. The analysis is effected in acuvette, with the attendant sensitivity allowed by analysis in acuvette. Sorting occurs in the steam in air, allowing sorting in theconventional manner. The relatively low velocity of the core stream inthe analysis region is advantageous for analysis of targets. The streamis then accelerated by the nozzle, to allow for high speed sorting.These advantages present a considerable cost savings due to both timesaved as well as skill required to use the system. This system alsosignificantly improves the depth and sensitivity of analysis and sortingperformance.

What is claimed is:
 1. A flow cell for use with a flow cytometercomprising: a flow cell body; a sample delivery tube extending into saidflow cell body; at least one sheath flow port on said flow cell body,said at least one port allowing introduction of a flow of sheath flowliquid through said flow cell body; a cuvette joined to the flow cellbody; a channel extending through the cuvette, wherein liquid from saidsample delivery tube and said at least one sheath flow port flows intosaid channel; and a removable nozzle at a terminus of said channel, saidnozzle held in a registered position on said flow cell, said registeredposition at a defined three dimensional position and at a registeredrotational orientation.
 2. The flow cell of claim 1, further including adroplet generator associated with said flow cell, said droplet generatorallowing production of droplets as a stream exits the nozzle.
 3. Theflow cell of claim 2, wherein said droplet generator is located withinsaid flow cell body such that droplet generation oscillations areimparted to flow fluid before said fluid enters the channel.
 4. The flowcell of claim 3, wherein said droplet generator includes a piston. 5.The flow cell of claim 2, wherein said droplet generator is a vibratingelement.
 6. The flow cell of claim 5, wherein said vibrating elementvibrates one of a group consisting of the nozzle, the cuvette, and theflow cell body.
 7. The flow cell of claim 1, wherein said removablenozzle is positioned on a removable nozzle key.
 8. The flow cell ofclaim 7, wherein said nozzle key includes a biasing spring on one sideof said key.
 9. The flow cell of claim 7, wherein said nozzle keyincludes an o-ring surrounding the nozzle.
 10. The flow cell of claim 1,wherein said cuvette has sidewalls that extend downward from a locationof said terminus of said channel.
 11. The flow cell of claim 1, whereinsaid channel has a rectangular cross section.
 12. The flow cell of claim1, wherein said channel has a length sufficiently long so that liquidflowing through said channel has fully developed flow when said fluidreaches an illumination location on said channel.
 13. The flow cell ofclaim 1, wherein when said nozzle is inserted into said registeredlocation, a nozzle opening of the nozzle is off center from a centeredposition within the channel.
 14. A flow cytometer system comprising: aflow cell body; a sample delivery tube extending into said flow cellbody; at least one sheath flow port on said flow cell body, said atleast one port allowing introduction of a flow of sheath flow liquidthrough said flow cell body; a cuvette joined to the flow cell body; achannel extending through the cuvette, said channel joined to said flowcell body such that liquid from said sample delivery tube and said atleast one sheath flow port flows into said channel; a removable nozzleat a terminus of said channel, said nozzle held in a registeredposition, said registered position at a defined three-dimensionalposition and at a registered rotational orientation; illumination opticswhich focus and direct illumination light into said flow cell at anillumination region of said flow cell; and light collection optics whichcollect light produced from targets in the channel of the flow cell andtransmit collected light to detection optics.
 15. The system of claim14, further including a droplet generator associated with said flowcell, said droplet generator allowing production of droplets as a streamexits the nozzle.
 16. The system of claim 15, wherein said dropletgenerator is located within said flow cell body such that dropletgeneration oscillations are imparted to flow fluid before said fluidenters the channel.
 17. The system of claim 16, wherein said dropletgenerator includes a piston.
 18. The system of claim 15, wherein saiddroplet generator is a vibrating element.
 19. The system of claim 18,wherein said vibrating element vibrates one of a group consisting of thenozzle, the cuvette, and the flow cell body.
 20. The system of claim 14,wherein said removable nozzle is positioned on a removable nozzle key.21. The system of claim 20, wherein said nozzle key includes a biasingspring on one side of said key.
 22. The system of claim 20, wherein saidnozzle key includes an o-ring surrounding the nozzle.
 23. The system ofclaim 14, wherein said cuvette has sidewalls that extend downward from alocation of said terminus of said channel.
 24. The system of claim 14,wherein said channel has a rectangular cross section.
 25. The system ofclaim 14, wherein said channel has a length sufficiently long so thatliquid flowing through said channel has fully developed flow when saidfluid reaches the illumination region.
 26. The system of claim 14,wherein when said nozzle is inserted into said registered location, anozzle opening of the nozzle is off center from a centered positionwithin the channel.
 27. The system of claim 14, further including alight transmissive coupling joining the cuvette with a light collectionlens, said lens being an element of the light collection optics.
 28. Thesystem of claim 24, wherein said channel is rectangular and has ashorter cross-sectional side and a longer cross-sectional side, whereinsaid shorter cross-sectional side faces a first direction in which lightis directed by the illumination optics and the longer cross-sectionalside faces a second direction from which emitted light is collected bythe light collection optics.
 29. A sorting flow cytometer componentincluding a flow cell and light collection optics comprising: a flowcell body; a sample delivery tube extending into said flow cell body; atleast one sheath flow port on said flow cell body, said at least oneport allowing introduction of sheath flow liquid into said flow cellbody; a cuvette; a channel extending through the cuvette, wherein liquidflowing from said sample delivery tube and said at least one sheath flowport flows into said channel as a flow stream, wherein said channel hasan illumination region, wherein light from an illumination source of aflow cytometer system illuminates liquid flowing through the channel,wherein emission light produced by said illumination light passes fromsaid channel into an optically transmissive material of the cuvettesurrounding said channel; a nozzle at a terminal end of said channel; adroplet generator associated with said flow cell body, said dropletgenerator allowing production of droplets from a stream exiting saidnozzle; a light collection lens; and a coupling material joining saidlight collection lens to said optically transmissive material, saidcoupling material allowing emission light to pass from said opticallytransmissive material into said light collection lens without atransition into air.
 30. The component of claim 29, wherein said nozzleis a removable nozzle, said nozzle held in a registered position saidregistered position registered in three dimension and rotationalorientation.
 31. The component of claim 29, wherein said cuvette hassidewalls that extend downward from a location of said terminus of saidchannel.
 32. The component of claim 29, wherein said channel has arectangular cross section.
 33. The component of claim 29, wherein saiddroplet generator is positioned within the flow cell body, said dropletgenerator producing an oscillating pressure wave in sheath fluid flowingthrough said flow cell body.
 34. The component of claim 29, wherein saidcoupling material is a gel material.
 35. The component of claim 29,wherein said channel has a length sufficiently long so that liquidflowing through said channel has fully developed flow when said fluidreaches the illumination region.
 36. The component of claim 29, whereinwhen said nozzle is inserted into a said registered position on saidflow cell, a nozzle opening of said nozzle is off center from a crosssectional center position within the channel.
 37. A sorting flowcytometer system comprising: a flow cell body; a sample delivery tubeextending into the flow cell body; at least one sheath flow port on saidflow cell body, said at least one port allowing introduction of sheathflow liquid through said flow cell body; a droplet generator associatedwith said flow cell body, said droplet generator allowing production ofdroplets from a stream exiting said nozzle; a cuvette; a channelextending through said cuvette, wherein liquid flowing from said sampledelivery tube and said at least one sheath flow port flows into saidchannel, wherein said channel has an illumination region, wherein lightfrom an illumination source of the flow cytometer system may illuminateliquid flowing through the channel at the illumination region, whereinemission light produced by said illumination light passes from saidchannel into an optically transmissive material surrounding saidchannel; a nozzle at a terminal end of said channel; illumination opticsthat focus and direct illumination light into said channel at theillumination region; a light collection lens; and a coupling materialjoining said light collection lens to said optically transmissivematerial surrounding the channel, said coupling material allowingemission light to pass from said optically transmissive material intosaid light collection lens without a transition into air.
 38. The systemof claim 37, wherein said nozzle is a removable nozzle, said nozzle heldin a registered position said registered position at a definedthree-dimensional position and at a registered rotational orientation.39. The system of claim 37, wherein said cuvette has sidewalls thatextend downward from a location of said terminus of said channel. 40.The system of claim 37, wherein said channel has a rectangular crosssection.
 41. The system of claim 37, wherein said droplet generator ispositioned within the flow cell body, said droplet generator producingan oscillating pressure wave in sheath fluid flowing through said flowcell body.
 42. The system of claim 37, wherein said coupling material isa gel material.
 43. The system of claim 37, wherein said channel has alength sufficiently long so that liquid flowing through said channel hasfully developed flow when said fluid reaches the illumination region.44. The system of claim 37, wherein when said nozzle is inserted into asaid registered position on said flow cell, a nozzle opening of saidnozzle is off center from a cross sectional center position within thechannel.
 45. A flow cell for use with a sorting flow cytometercomprising: a flow cell body; a sample delivery tube extending into saidflow cell body; at least one sheath flow port on said flow cell body,said at least one sheath flow port allowing introduction of a flow ofsheath flow liquid through said flow cell body; a cuvette; a channelextending through the cuvette, wherein liquid flowing from said sampledelivery tube and said at least one sheath flow port flow into saidchannel; a nozzle at a terminal end of the channel; and a dropletgenerator associated with said flow cell body, said droplet generatorallowing production of droplets in said flow stream, said dropletgenerator imparting an oscillating force to fluid flowing into said flowcell body without direct vibration of the flow cell body or the nozzle.46. The flow cell of claim 45, wherein said cuvette has sidewalls thatextend downward from a location of the terminal end of the channel. 47.The flow cell of claim 45, wherein said channel has a rectangular crosssection.
 48. The flow cell of claim 45, wherein said channel has alength sufficiently long so that liquid flowing through said channel hasfully developed flow when said fluid reaches the nozzle.
 49. A flowcytometer system comprising: a flow cell body; a sample delivery tubeextending into said flow cell body; at least one sheath flow port onsaid flow cell body, said at least one port allowing introduction of aflow of sheath flow liquid through said flow cell body; a cuvette; achannel extending a length through the cuvette, wherein liquid flowingfrom said sample delivery tube and said at least one sheath flow portflow into said channel; a nozzle at a terminal end of the channel; adroplet generator associated with said flow cell body, said dropletgenerator allowing production of droplets in said flow stream, saiddroplet generator imparting an oscillation to fluid flowing into saidflow cell body without direct vibration of the flow cell body or thenozzle; illumination optics which focus and direct illumination lightinto said flow cell at an illumination region of said channel; and alight collection optics that collect emitted light from said channel.50. The system of claim 49, further including a light transmissivecoupling joining the cuvette with a light collection lens, said lensbeing an element of the light collection optics.
 51. The system of claim49, wherein said cuvette has sidewalls that extend downward from alocation of said terminal end of said channel.
 52. The system of claim49, wherein said channel has a rectangular cross section.
 53. The systemof claim 52, wherein the rectangular cross section has a shorter crosssectional side and a longer cross-sectional side, wherein said shortercross sectional side faces a first direction in which light is directedby the illumination optics and the longer cross sectional side faces asecond direction from which emitted light is collected by the lightcollection optics.
 54. The system of claim 49, wherein said channel hasa length sufficiently long so that liquid flowing through said channelhas fully developed flow when said fluid reaches the nozzle.
 55. A flowcell for use with a sorting flow cytometer comprising: a flow cell body;a sample delivery tube extending into said flow cell body; at least onesheath flow port on said flow cell body, said at least one port allowingintroduction of a flow of sheath flow liquid; a cuvette joined to saidflow cell body; a rectangular channel extending a length through saidcuvette, wherein liquid flowing from said sample delivery tube and saidat least one sheath flow port flow into said channel; a dropletgenerator connected to said flow cell, said droplet generator allowingproduction of droplets from a liquid stream exiting said nozzle; and anozzle at a terminus of said channel, wherein the cuvette has sidewallsthat extend below said terminus of the channel, said sidewalls beingoptically transmissive such that light emitted from liquid in thechannel may be transmitted through said sidewalls to light collectionoptics.
 56. The flow cell of claim 55, wherein said channel has a lengthsufficiently long so that liquid flowing through said channel has fullydeveloped flow when said fluid reaches the nozzle.
 57. The flow cell ofclaim 56, wherein the nozzle is held at a registered position saidregistered position at a defined three-dimensional position and at aregistered rotational orientation.
 58. The flow cell of claim 55,wherein said nozzle is a removable nozzle, said nozzle held in aregistered position, said registered position at a definedthree-dimensional position and at a registered rotational orientation.59. A sorting flow cytometer system comprising: a flow cell body; asample delivery tube extending into said flow cell body; at least onesheath flow port on said flow cell body, said at least one port allowingintroduction of a flow of sheath flow liquid; a droplet generatorconnected to said flow cell, said droplet generator allowing productionof droplets in said flow stream; a cuvette joined to said flow cellbody, said cuvette having; a rectangular channel extending through saidcuvette to a terminus of said channel, wherein liquid flowing from saidsample delivery tube and said at least one sheath flow port flow intosaid channel; a nozzle at the terminus of said channel, wherein thecuvette has sidewalls that extend below said terminus of the channel,said sidewalls being optically transmissive such that light emitted fromliquid in the channel may be transmitted through said sidewalls to lightcollection optics; an illumination input optics which focuses anddirects illumination light into said flow cell at an illumination regionof said channel; and light collections optics which collect lightproduced from targets in the channel of the flow cell and transmitcollected light to detection optics.
 60. The system of claim 59, furtherincluding a light transmissive coupling joining the cuvette with a lightcollection lens, said lens being an element of the light collectionoptics.
 61. The system of claim 59, wherein said channel has a lengthsufficiently long so that liquid flowing through said channel has fullydeveloped flow when said fluid reaches the nozzle.
 62. The system ofclaim 61, wherein the nozzle is held at a registered position saidregistered position at a defined three-dimensional position and at aregistered rotational orientation.
 63. The system of claim 59, whereinsaid nozzle is a removable nozzle, said nozzle held in a registeredposition, said registered position at a defined three-dimensionalposition and at a registered rotational orientation.
 64. A flow cell foruse with a flow cytometer comprising: a flow cell body; a sampledelivery tube extending into said flow cell body; at least one sheathflow port on said flow cell body, said at least one port allowingintroduction of a flow of sheath flow liquid; a cuvette; a channelextending through the cuvette and having a longitudinal axis, saidchannel joined to said flow cell body such that liquid from said sampledelivery tube and said at least one sheath flow port flow into saidchannel; and a removable nozzle at a terminus of said channel, saidnozzle held in a registered position such that at said registeredposition a nozzle opening of said nozzle is off-center from thelongitudinal axis of the channel.
 65. The flow cell of claim 64, whereinsaid cuvette has sidewalls that extend downward from a location of aterminal end of said channel.
 66. The flow cell of claim 64, whereinsaid channel has a rectangular cross section.
 67. The flow cell of claim64 wherein said channel has a length sufficient such that flow of fluidthrough the channel is fully developed at the nozzle location.
 68. Aflow cytometer system comprising: a flow cell body; a sample deliverytube extending into said flow cell body; at least one sheath flow porton said flow cell body, said port allowing introduction of a flow ofsheath flow liquid; a cuvette joined to said flow cell body, saidcuvette having; a rectangular channel extending a length through saidcuvette to a terminus of said channel, wherein liquid flowing from saidsample delivery tube and said at least one sheath flow port flow intosaid channel; a nozzle at the terminus of said channel, wherein thecuvette has sidewalls that extend below said terminus of the channel,said sidewalls being optically transmissive such that light emitted fromliquid in the channel may be transmitted through said sidewalls to lightcollection optics; an illumination input optics which focuses anddirects illumination light into said flow cell at an illumination regionof said channel; and light collections optics which collect lightproduced from targets inn the channel of the flow cell and transmitcollected light to detection optics.
 69. The system of claim 68, whereinsaid channel has a length sufficiently long so that liquid flowingthrough said channel has fully developed flow when said fluid reachesthe nozzle.